Field of the Invention: The present invention is an optical method for use in the field of blood clotting, in particular for the study of drug effects on clotting, the detection of clotting abnormalities, and other disease states.
The proper clotting of blood is clearly essential to health. Although insufficient clotting that leads to bleeding is a serious problem for some individuals, an increased tendency to clot that results in stroke, myocardial infarction, or deep vein thrombosis affects a vastly greater number of people. To ward off these thrombotic events, many people take a daily dose of aspirin. Multiple strokes can cause dementia, usually called vascular dementia in this case. The more common form of dementia, Alzheimer's disease, has some points of overlap with vascular disease. For example, certain types of cholesterol-carrying proteins can increase the risk of both myocardial infarction and Alzheimer's disease. Furthermore, people who take ibuprofen have a lower risk of developing Alzheimer's disease than those who do not.
Ibuprofen and aspirin are both members of the same family of drugs, namely, non-steroidal anti-inflammatory drugs (NSAIDs). Most NSAIDs block the conversion of arachidonic acid into its active metabolites. There are three major enzymes that convert arachidonic acid: lipoxygenase, cyclooxygenase-1 (COX1), and cyclooxygenase-2 (COX2). The primary effect of aspirin in preventing thrombotic events is through the inhibition of COX1; the effect of ibuprofen in the prevention of Alzheimer's disease probably does not involve any of these three pathways.
NSAIDs are also used to treat various inflammatory conditions, such as arthritis. In order to maximize therapy for these inflammatory conditions without affecting blood clotting, NSAIDs that specifically inhibit COX2 were developed. Rofecoxib and celecoxib are two commonly prescribed COX2-specific inhibitors. Neither rofecoxib nor celecoxib has any effect on clotting as measured by routine techniques, and both may be administered to people taking anticoagulant drugs like warfarin. It was, therefore, surprising when a study comparing rofecoxib and naproxen (another NSAID) reported a higher incidence of thrombotic events in the rofecoxib-treated arm (Bombardier et al., N.Eng.J.Med. 2000;343:1520–1528). One explanation held that naproxen somehow protected against thrombotic events, as does aspirin (Bombardier et al., N.Eng.J.Med. 2000;343:1520–1528); another explanation is that rofecoxib promoted coagulation (Mukherjee et al., JAMA 2001 ;286:954–959). Because no routine test of coagulation detects an effect of COX2 inhibitors, there is a need for a method that can show such an effect in order to investigate the reason for the difference between rofecoxib and naproxen.
As mentioned above, there are many points of overlap between Alzheimer's disease and thrombotic vascular disease. Alzheimer's disease involves the abnormal deposition of material in the brain. An important component of this abnormal material consists of fragments of protein cleaved from a larger protein called amyloid-precursor protein (APP). The enzyme which cleaves APP to form these fragments is called γ-secretase; the bulk of the evidence favors γ-secretase as the site of action of ibuprofen in the prevention of Alzheimer's disease. APP is also found in blood platelets and is normally cleaved during blood clotting. The cleavage of APP in platelets is abnormal in patients with Alzheimer's disease. Furthermore, abnormalities in the red cell are found in Alzheimer's disease (Greco et al., Arch. Pathol. Lab. Med. 2000;124:1141–1146). The present invention grew out of attempts to improve the method that used conformational changes in erythrocyte band 3 to diagnose Alzheimer's disease (U.S. Pat. No. 6,030,768). Although numerous abnormalities have been described in platelets and red cells, none is sufficiently powerful to serve as a diagnostic test for Alzheimer's disease. Because Alzheimer's disease has an insidious onset, there is a need for a test that may detect abnormalities in APP cleavage before the onset of dementia. Furthermore, if this test also detected effects of NSAIDs, it would be useful in gauging the response to prophylactic therapy.
Description of Related Art: There are numerous tests to study blood clotting. Many use the time-to-clot as the endpoint and are insensitive to subtle changes in blood, e.g., the Lee-White clotting time, prothrombin time, partial thromboplastin time, bleeding time. The time-to-clot may be measured optically, mechanically, or electrically, which accounts for most of the instrumental differences among the various methods. Levels of individual clotting factors can be measured, but this is usually done to diagnose a deficiency, not as a routine test of clotting.
Many optical techniques have been applied to blood. When applied to coagulation, the decrease in light transmission as platelet-poor plasma clots is often used. In studying the function of isolated platelets, light scattered at 90° is measured in response to divers agents. As mentioned above, none of these methods can detect an effect of COX2 inhibitors on clotting.
Description of Prior Art Optical: techniques have also been applied to blood rheology, that is, the flow of blood. There are several methods that monitor optical changes in reflected light at various flow rates as the red cells migrate centrally along the axis of flow. If the anticoagulant is reversed, the increase in viscosity that accompanies coagulation may be tracked (Riha et al., Clin. Hemorheol. Mircocirc. 1997;17:341–346). In one special case, the optical changes that accompany clotting resemble those of the present invention (See FIG. 2, Riha et al., Clin. Hemorheol. Mircocirc. 1997;17:341–346). The features of the present invention that distinguish it from and improve upon the rheological technique are discussed in the DETAILED DESCRIPTION OF THE INVENTION. Briefly, the present invention differs from the rheological technique in being stationary, in its method of analysis, and use of broadband illumination.